Biphasic activation of PKBα/Akt in platelets -: Evidence for stimulation both by phosphatidylinositol 3,4-bisphosphate, produced via a novel pathway, and by phosphatidylinositol 3,4,5-trisphosphate

Stimulation of platelet thrombin receptors or protein kinase C causes fibrinogen-dependent aggregation that is a function of integrin alpha(IIb)beta(3) activation. Such platelets rapidly and transiently form phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) and a small amount of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P-2). After aggregation, a larger amount of PtdIns(3,4)P-2 is generated. We report that this latter PtdIns(3,4)P-2 arises largely through wortmannin-inhibitable generation of PtdIns3P and then phosphorylation by PtdIns3P 4-kinase (PtdIns3P 4-K), a novel pathway apparently contingent upon the activation of the Ca2+-dependent protease calpain. Elevation of cytosolic Ca2+ by ionophore, without integrin/ligand binding, is insufficient to activate the pathway. PtdIns3P 4-K is not the recently described "PIP5KII alpha." Cytoskeletal activities of phosphatidylinositol 3-kinase and PtdIns3P 4-K increase after aggregation. Prior to aggregation, PtdIns3P 4-K can be regulated negatively by the beta gamma subunit of heterotrimeric GTP-binding protein. After aggregation, PtdIns3P 4-K calpain-dependently loses its susceptibility to G beta gamma and is, in addition, activated. Both PtdIns(3,4,5)P-3 and PtdIns(3, 4)P-2 have been shown to stimulate PKB alpha/Akt phosphorylation and activation by phosphoinositide-dependent kinase 1. We find that activation of PKB alpha Akt in platelets is phosphorylation-dependent and biphasic; the initial phase is PtdIns(3,4,5)P-3-dependent and more efficient, whereas the second phase depends upon PtdIns(3,4)P-2 generated after aggregation. There is thus potential for both pre-and post aggregation-dependent signaling by PKB alpha Akt.